In recent years, the magnetic resonance imaging (MRI) has been developed rapidly and become one of the most important techniques for diagnosing diseases. In order to increase sensitivity and accuracy, it is very important to develop a safe, stable, and high relaxivity MRI contrast agent. The general metal ions for MRI contrast agents used include Mn2+, Fe3+, and Gd3+, wherein among the metal ions, Gd3+ is the most commonly used one due to its greatest magnetic moment (μM) resulting form the seven unpaired electrons thereof. However, such a cation, Gd3+, is not suitable for use alone as a MRI contrast agent due to its greatest toxicity in animal bodies. Therefore, it is necessary to use a ligand to chelate Gd3+ to form a stable metal complex to inhibit the toxicity thereof.
To design a new contrast agent for MRI, the stability of the metal complex is the main concern. The contrast agent should be effective during the period of time from injecting it to the body to excreting it from the body. Therefore, stability is required for this residence time. Three factors should be considered to determine the stability of a gadolinium complex in vivo, i.e. the thermodynamic stability constant, the conditional stability constant, and the selectivity constant of the gadolinium complex (Cacheris et al, 1990, Magn. Reson. Imag., vol. 8, p 467), wherein the thermodynamic stability constant represents an affinity of the fully deprotonated ligand and the gadolinium (III), and the conditional stability constant represents a stability constant for the gadolinium complex at the physiological pH in vivo. Since there are other metal ions, such as the calcium ion, the zinc ion, the copper ion, and the iron ion, in the organisms, these mentioned ions would compete with the gadolinium (III) for coordinating with the ligand. Accordingly, if the selectivity of the ligand for the gadolinium complex is lower than the others, the gadolinium (III) will release from the gadolinium complex.
The relaxivity of the metal complex is also an important consideration for designing an MRI contrast agent. Generally, the factors affecting the relaxivity are represented by the following equation:r1≈q(μeff)2τc/r6 wherein q represents the hydration number (the number of the water molecules in the inner-sphere); μeff represents the effective magnetic distance of the metal ion (as to the gadolinium ion, μeff thereof is 0.94 Bohr Magneton); τc represents the correlation time of a paramagnetic material in a fixed magnetic field; and r represents the distance between the metal ion and the protons of the water molecules in the inner-sphere (as to the Gd3+OH2 system, r thereof is 2.50±0.04 Å, e.g. Schauer C. K. A. et al, 1989, J. Chem. Soc., Dalton Trans, p 185). For the gadolinium complexes with similar functional groups, since the values of μeff and r could be considered as constants, the main factors for the relaxivity are q and τc.
Mainly the correlation time (τc) is influenced by the three factors mentioned below: (1) molecular rotational correlation time, τr; (2) the electron longitudinal and transverse spin relaxation time, T1, 2e; and (3) the inner-sphere water residence lifetime, τm or exchange rate, τm−1=kex(Tóth, E. et al, 2001, Coord. Chem. Rev, Vol. 216-217, p 363). The relation is shown by the equation:τc−1=τr−1+Tie−1+τm−1 i=1,2wherein the relaxivity would be a maximum if the value of correlation time τc equals to the reciprocal value of the proton Larmor frequency. So it is inferred that the optimum value of τc is 7.4 ns while the magnetic field strength is 0.5 T (21 MHz 1H frequency) and 2.5 ns while the magnetic field strength is 1.5 T (64.5 MHz).
It is suggested that the longitudinal relaxivity mainly depends on the longitudinal relaxation of bound solvent molecule, T1m and τm (Luz et al., 1964, Chem. Phys. Vol. 40, p 2686). The relation is shown by the following equation:
      r    1    =            1              T        1              =                  qP        m                              T                      1            ⁢            m                          +                  τ          m                    where Pm is the mole fraction of the bound solvent molecules. From the above-mentioned equation, it appears that if the exchange rate of water molecule is very fast, i.e. τm<<T1m, then r1 mainly depends on the relaxivity of the bound solvent molecule (1/T1m). Therefore, in order to achieve a higher relaxation rate, a very small value of τm of the gadolinium complex is usually required. However, if τm is too small, then T1m would be influenced by τm, so a higher relaxivity is unachievable even though the value of τm is reduced unlimitedly. Through a theoretical simulation for the influence of T1e, τr, and τm on relaxivity, it is obtained that the optimum value of τm is 10 ns under the condition of simulating q=1 and r=3.1 Å while the magnetic field strengths are respectively 0.5 T and 1.5 T, i.e. the most commonly used two magnetic field strengths in clinical (Caravan et al., 1999, Chem. Rev. Vol. 99, p 2293).
Generally speaking, the augmentation of the magnetic field strength would lead to an increase of T1e. T1e has a great influence on the relaxivity in a magnetic field of 0.5 T. However, T1e does not have a very obvious influence on the relaxivity in a magnetic field of 1.5 T. That is, the relaxivity is only influenced by τr and τm while the magnetic field strength is higher. The optimum value of τm is about 10 ns as mentioned in the previous paragraph, whereas the optimum value of τr is about 20 ns. The longitudinal relaxation rates of all available MRI contrast agents in the market now are somewhat lower than their theoretical maximum values. This is mainly because the molecular rotational correlation time values of these contrast agents are small.
The new generation of MRI contrast agents relates to a conjugation of the metal complex of small molecules (with a low molecular weight), such as [Gd(DOTA)]2− or [Gd(DOTA)]− (Brasch, 1991, Magn. Reson. Med., Vol. 22, p 282), with something with a high molecular weight, so as to adjust their biophysical and pharmacological properties. From the view of biophysics, the molecular rotational correlation time of the contrast agent is lowered by means of the combination of the gadolinium complex of small molecules and the polymeric materials, and the relaxivity is thus increased. Besides, if the gadolinium complexes are combined with tissue-specific targeting moieties, these polymeric conjugations will bring the gadolinium complexes to receptors at low concentration by carriers, so that the receptors would be observed in MRI. Besides, because the molecules of the conjugations with high molecular weights are bigger, they would stay in vascular for a longer time, and thus they are suitable for use in the blood pool imaging.
Misckei K. H. et al. have disclosed the complex [Gd(DTPA)(H2O)]2− with a τm equal to 303 ns, which is much greater than the optimum τm (Micskei K. H. et al., 1993, Inorg. Chem., Vol. 32, p 3844). Laurent S. E. et al. have disclosed the complex [Gd(DTPA)(H2O)]2− with a τr equal to 59 ps, which is much smaller than the optimum τr (Laurent S. E. et al., 2000, Helv. Chim. Acta, Vol. 83, p 394). Hence, the scientists in the field are enthusiastically seeking for a metal complex with the high water exchange rate and the low rotational correlation time, whereby increasing of the relaxivity r1 for the metal complex is desired.
A lot of efforts have been spent on trying to lower the molecular rotational correlation time of the contrast agent in order to increase the relaxivity thereof. For example, the molecular rotational correlation time is lowered by substituting one of the carboxylate in the structure of 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA) with larger functional groups containing benzenes. Because of the increase of the value of τr, the value of r1 climbs according to the increase of the molecular weights of the ligands (Lauffer et al., 1987, Chem. Rev., Vol. 87, p 901; Aime et al., 1992, Inorg. Chem., Vol. 31, p 2422).
There are many methods developed for linking the gadolinium complex to a high molecular weight residue (Brinkley, 1992, Bioconjugate Chem., Vol. 3, p 2), wherein the acylation, the alkylation, the formation of ureas, and the reduction of amination are popular applied. The most frequently used reagents to be combined with a high molecular weight moiety include DTPA, the derivatives thereof, and DTPA-dianhydride. By the reaction of the primary amine in the high molecular weight moiety with DTPA, the derivatives thereof, or DTPA-dianhydride, the ligands would be combined therewith. Sieving et al. (1990, Bioconjugate Chem., Vol. 1, p 65) disclose the reaction of polylysines with variant molecular weights with DTPA-dianhydride and the derivatives of DTPA. However, the cross-linking always happens very easily during the combination of DTPA-dianhydrare and proteins. Therefore, by the reaction of N-hydroxysuccinimide with DTPA to form a N-hydroxysuccinic ester, the crossing-linking is evitable (Spanoghe et al., 1992, Magn. Reson. Imaging, Vol. 10, p 913). Paxton et al. (1985, Cancer Res., Vol. 45, p 5694) disclose that [DTPA-(N-hydroxysuccinic ester)] would form a covalent binding not only with the protein but also with the monoclonal antibody, and by this carrier the contrast agent would be brought to the anticarcinoembryonic antigen. However, this synthetic method also leads to the production of peptide bonds, so that the binding ability of this ligand to the gadolinium complex is weakened. In order to overcome this problem, Aime et al. (1999, Bioconjugate Chem., Vol. 10, p 192) disclose a method in which the covalent binding is formed between 1,4,7-trikis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A) and proteins with a linker of 3,4-diethoxycyclobut-3-ene-1,2-dione,squarate. Through this method, the stability of the metal complex is enhanced, and the relaxivity of the gadolinium complex is also increased due to the augment of the molecular weight. In addition, a specific bioactive is achieved via the formation of the covalent binding with the protein.
Because the concentration at which MRI contrast agent is capable of targeting lesions (such as receptors or antigens) falls in a nanomolar level, which is too low for the receptor-induced magnetization enhancement (RIME) to be used in MRI, during the recent years scientists have tried to use the enzyme to activate the gadolinium complex so as to increase the concentration of the contrast agent at the targeted location approximately. Through the method, the relaxation rate of the metal complex is increased, and the target-to-background ratio is also enhanced. Furthermore, 4,7,10-tri(aceticacid)-1-(2-β-galactopyranosylethoxy)-1,4,7,10-tetraazacyclododecane gadolinium (III), Egad, is synthesized by connecting one of the functional groups to the macrocyclic ligand (Moats et al., 1997, Chem. Int. Engl., Vol. 36, p 726). In this case, because Egad is in the form of 9-coordinate, the number of inner space water molecules of Egad is 0.7. That is to say, when Egad enters the organism and meets the β-galactosidase (β-gal), the o-nitrophenyl-β-galactopyranoside in the Egad would be hydrolyzed and removed so as to be bound with 1.2 inner space water molecules, and the MRI signals are thus enchanced.
The Pro-RIME with an increased receptor-induced magnetization is also synthesized (Nivorozhkin et al., 2001, Angew. Chem. Int. Ed., Vol. 15, p 2903). This reagent is composed of: (1) a masking group consisting of three lysine residues, (2) an HSA binding site, (3) a glycine linker, and (4) a signal generation group. The mechanism of action involves that the degradation of lysines in the outermost space is easily achieved by the human carboxypeptidase B thrombin-activatable fibrinolysis inhibitor (TAFI). Once the degradation of the three lysine residues is completed, the exposed lipid soluble aromatics would produce a great binding ability with HSA, and thus a higher relaxation rate is achieved.
A novel MRI contrast agent [Gd(BOPTA)]2−, which is capable of forming a stable complex with its relaxivity valued 4.39 mM−1 s−1 higher than that of [Gd(DTPA)]2− (3.77 mM−1 s−1), has been disclosed by Fulvio U. et al. In that research, it is found that the inner-sphere water residence lifetime, τm, is 289 ns, which is much higher than the theoretical optimum value, 10 ns. In addition, the relaxivity valued 33.0 mM−1 s−1 for the combination of the mentioned agent [Gd(DTPA)]2− with human serum albumin is also lower than that of MS-325 (47.0 mM−1 s−1) (Fulvio U. et al., 1995, Inorg. Chem., Vol. 34, p 633).
The process for the preparation of [Gd(BOPTA)]2− has been disclosed in the U.S. Pat. No. 6,162,947 by Marina A. et al. In the mentioned patent, the preparation processes and the advantages of [Gd(BOPTA)]2− are disclosed in detail, and a best process for preparing [Gd(BOPTA)]2− is summarized therein.
The binding constants with HSA, KA of [cis-Gd(DOTA-BOM2)]−, [trans-Gd(DOTA-BOM2)]−, [Gd(DOTA-BOM3)]−, [Gd(DTPA-BOM3)]2− and MS-325 are 3.2±0.4×102M−1, 3.6±0.4×102M−1, 1.7±0.1×103 M−1, 4.0±0.3×104 M−1 and 3.0±0.2×104 M−1, respectively; the bound relaxivities, r1b, of the above-mentioned complexes are 35.7 mM−1 s−1, 44.2 mM−1 s−1, 53.2 mM−1 s−1, 44.0 mM−1 s−1 and 47.0 mM−1 s−1, respectively (Silvio A. et al., 1996, J. Biol. Inorg. Chem., Vol. 1, p 312; 1999, J. Biol. Inorg. Chem., Vol. 4, p 766). In the mentioned researches, it is found that the binding constant and the bound relaxivity are increased with the number of the benzyloxymethyl group of the complex.
A novel MRI contrast agent [Gd(AAZTA)]-(1,4-bis(t-butoxycarbonylmethyl)-6-[bis(t-butoxycarbonylmethyl)])amino-6-methylperhydro-1,4-diazepine, AAZTA) synthesized by Silvio A. et al., has three nitrogen and four carboxylic group included, and is capable of coordinating with gadolinium (III) for forming a complex with seven coordinates. The occurrence of q=2 (in which q is the number of the water molecules) has been assessed by measuring Dy (III) induced 17O NMR water shift (which is performed by the d.i.s. measurement). The relaxivity of [Gd(AAZTA)]− is 7.1 mM−1 s−1 at 20 MHz and 298 K, which is obviously higher than the relaxivity valued 3.89 mM−1 s−1 of [Gd(DPTA)]2−. As to the thermodynamic stability, a log KGdL value of 19.26 for [Gd(DPTA)]2− complex was obtained, where such a value is slightly smaller than [Gd(DTPA)]2− (logKGdL=22.46) but is significantly higher than that of [Gd(DTPA-BMA)] (logKGdL=16.85). According to the mentioned data, it is concluded by the researcher that [Gd(AAZTA)]− complex with high stability is qualified for serving as an MRI contrast agent. In addition, τr=74 ps and τm=90 ns for [Gd(AAZTA)]− system have been obtained by NMRD data. Moreover, a high relaxivity value of 100 mM−1 s−1 has been calculated by simulating the NMRD profile in the situation that τr is increased to 30 ns.
Currently, the six MRI contrast agents approved by FDA for clinical usage in intravenous injection include [Gd(DTPA)]2− (gadopentetate dimeglumine), [Gd(DOTA)]− (gadoterate megulumine), [Gd(DTPA-BMA)] (bis-methylamide gadodiamide injection), [Gd(HP-DO3A)] (gadoteridol), [Gd(BOPTA)]2− (gadobenate dimeglumine), and MnDPDP (Teslascan). The above-mentioned agents are extracellular agents, wherein [Gd(DTPA-BMA)] and [Gd(HP-DO3A)] are nonionic contrast agents; [Gd(DTPA)]2−, [Gd(DOTA)]− and MnDPDP are ionic contrast agents; [Gd(DOTA)]− and [Gd(HP-DO3A)] have macrocyclic structure; and MnDPDP, [Gd(DTPA)]2− and [Gd(DTPA-BMA)] have the open-chained structures.
A derivative of DTPA, 3,6,10-tri-(carboxymethyl)-3,6,10-triazadodecanedioic acid (TTDA), has been synthesized and the physical and chemical properties of the metal complexes of Gd3+, Zn2+, Ca2+ and Cu2+ formed therewith are also studied in detail by Wang et al. The studies show that [Gd(TTDA)]2− has better physical and chemical properties than [Gd(DTPA)]2−, so it has a great potential to be an MRI contrast agent (Wang et al., 1998, J. Chem. Soc. Dalton Trans., p 4113-4118).
Based on the above, gadolinium (III) complexes with a high stability and a high relaxivity are the emphases of research during the recent years. It is important and useful to find better MRI contrast agents. Hence the present invention provides new metal complexes with potentiality and high stability as the MRI contrast agents.